PROJECT SUMMARY Trypanosoma brucei is a causative agent of both human and animal African trypanosomiasis, devastating diseases affecting sub-Saharan Africa. A dense variant surface glycoprotein (VSG) coat covers this extracellular parasite and effectively masks all other antigens on the cell surface. T. brucei evades elimination by the mammalian host's immune system through antigenic variation of its VSG coat: it ?switches? the expressed VSG, taking advantage of a genomic repertoire of ~2000 VSG-encoding genes. However, parasite populations in vivo express a diverse set of VSG-encoding genes. The number of expressed VSGs over 30 days of infection can approach one-third of the functional VSG repertoire within the parasite genome, which highlights an interesting question: if the existing genomic repertoire can be used up in a matter of months, how can an infection be sustained for years in the field? The goal of this application is to investigate the mechanisms by which T. brucei diversifies its genomic VSG repertoire in order to sustain infection. Preliminary data suggest that extravascular spaces may provide an important area for T. brucei to diversify its VSG archive in the absence of immune pressure. In the first aim of this proposal, the role of this extravascular niche in VSG diversification will be investigated. The second aim will take advantage of CRISPR/Cas9 technology combined with a novel high-throughput sequencing method to investigate the segmental gene conversion events resulting in novel ?mosaic? VSGs. In the third aim, the mutation of individual VSGs will be studied, in order to understand the mechanisms that govern the evolution of the VSG archive within the parasite genome over the course of infection. These studies will begin to elucidate the mechanisms by which T. brucei sustains a long- term infection in its host, which will be important for the development of new strategies for treatment of this neglected tropical disease. Three aims are proposed to accomplish these goals: 1) To elucidate the role of extravascular spaces in VSG diversification; 2) To identify the mechanisms responsible for the segmental gene conversion events that generate mosaic VSGs; 3) To quantify the rate of hypermutation of VSGs.